The goal of this proposal is to determine the mechanism of v-secretase-mediated processing of amyloid precursor protein (APR) and the role of the new ^-cleavage in the amyloidogenesis of Alzheimer's disease (AD). Accumulating evidence supports the amyloid hypothesis as being the predominant theory underlying the pathogenesis of Alzheimer's disease. Thus, the formation and accumulation of [unreadable]-amyloid peptide (A[unreadable]) in the brain is the central issue in AD research. We recently discovered a novel intracellular A[unreadable]46. According to the amyloid theory, the long Af3 is more amyloidogenic and pathogenic and, specifically, evidence suggests that the intracellular A[unreadable] plays a more important role in Alzheimer's disease. Thus, the discovery of the long A[unreadable]46 may provide a new target for diagnosis and treatment of Alzheimer's disease. The identification of A[unreadable]46 led to our discovery of a new ^-cleavage site in APR at A[unreadable]46, which happens to be the APP717 mutation site, suggesting that this AD-linked mutation may cause an increase in A[unreadable]42 by altering the ^-cleavage. We also detected the long sought after A[unreadable]49 produced by e-cleavage, and the detection of these intermediates enabled us to establish, for the first time, that the C-termini of A[unreadable] are generated by a series of sequential cleavages, namely e-cleavage at A[unreadable]49, ^-cleavage at A[unreadable]46, and y-cleavage at A[unreadable]40/42, commencing at the site closest to the membrane boundary and proceeding toward the site within the middle of the transmembrane domain of APR. More importantly, our finding that most of the known y-secretase inhibitors inhibit the formation of secreted A[unreadable]40 and A[unreadable]42, but cause an accumulation of their precursor, namely the long intracellular A[unreadable]46, provides information extremely important for the strategies to prevent and treat Alzheimer's disease by the design and use of y-secretase inhibitors. The discovery of the new [unreadable]-site and the detection of these intermediates open a new avenue for studying the mechanism of y-secretase-mediated APR processing and A[unreadable] formation. Based on our new findings and observations reported by previous studies, we hypothesize that: (I) three major cleavages, E-, [unreadable]-, and y- cleavages, are involved in the intramembrane processing of APP and also Notch 1;(II) AD-linked mutations in APR and presenilin cause increased A[unreadable]42 by causing a shift of the e-, [unreadable]-, and y-cleavage site(s);(III) in addition to the two aspartate residues, some of the threonine and serine residues in the transmembrane domains of presenilin may also be involved in the y-secretase-catalyzed multicleavages;(IV) both AICD and the GxxxG motif of APP may play a role in the interaction of APP with y-secretase;and (V) the upstream E- and ^-secretase occur mostly in the Golgi and trans-Golgi network (TGN), while the downstream y-secretase may also occur in the endosomal/lysosomal system in addition to TGN. The goal of this proposal will be achieved by the following Specific Aims: 1) Determine the significance of A[unreadable]46 in AD pathogenesis and the effects of AD-linked and systematically engineered mutations in APP and presenilins on the sequential E-, [unreadable]-, and y-cleavages and A[unreadable] production;2) Study comparatively the intramembranous processing of APP and Notch and determine whether some of the threonine and serine residues in the transmembrane domains of presenilin may also be involved in the y-secretase activity;and 3) Determine the cellular sites of e-, [unreadable]-, and y-cleavages and the roles of GxxxG motif and AICD of APP in the assembly of the y-secretase-substrate complex. P